All DeRiPRO solutions come in the form capable for RNA extraction; if DNA/protein extraction is desired, the solutions should be aliquoted to smaller and different containers for the purpose of DNA or protein extraction.
Prior to doing DNA extraction, 50 mg/ml of RNase must be added to the DRoP1 solution. KEEP AWAY FROM RNA WORK!
Incubate DRoP1 solution in 65°C for 15 minutes if precipitation occurs.
Prior to addition of DRoP1, samples should be treated with appropriate enzymes in ddh2O/ultrapure water (not in lysis buffer) if necessary (Gram-bacteria or yeast-like organisms) or being ground to powdery form using liquid nitrogen. Enzyme-treated Samples will then be pelleted (& washed a few times for protein extraction step) and dispersed by vortex or rough agitation.Samples will then be collected in 1.5ml tube (or bigger 15ml/50ml tube for larger volume sample).
1 For every 1ml or 0.19 samples, add 100–200ul of DRoP 1.
Protein Extraction Gentle inversions for mixing.
Lysis should immediately occur (cloudy sample should be cleared…difficult to see for powdery sample due to present of larger cell debris — prior to spinning), if not, incubate in 65°C for 5 minutes to assist lysis. (Spin at 13000xg for 5 minutes to remove large debris/impurity, collect 3. the supernatant.) Proceed to SDS-PAGE and immunodetection or e” Tagging protein purification procedure accordingly. (Larger volume of DRoP1 can be added if solution is too sticky)
DNA/RNA Extraction (3 Steps)
1 For every 1ml (liquid culture, spin down and pour off supernatant) or 0.19 (solid) samples, add 200ul of DRoP1.
Gentle inversions for mixing.
For plant or hard to lyse samples, incubate in 65°C for 5 minutes to assist lysis.
For DNA extraction only, incubate the clear solution in 37°C for 3 minutes.
For RNA extraction only, if degradation of RNA occurred possibly due to the RNase activity, proceed with all steps at 4°C.
For plasmid extraction, add DRoP Plasmid to DRoP1 at a ratio of 1:5 and mix by gentle inversion prior to use this to replace DRoP1 in steps.
2 Add 200ul of DRoP2, mix by gentle inversion and spin at 13,000xg for 3 minutes at room temperature to remove impurity.
Genomic DNA sample should be spun at 10000xg only.
Higher spin speed and longer spinning time is meant for impurity that is hard to stick to the bottom of the tube.
3 Pour off clear supernatant to new tube before adding 800ul of DRoP3, mix by gentle inversion and spin at 10,000xg – 16,000xg for 6 – 10 minutes at room temperature. Pour off supernatant and air dry for 5 – 15 minutes (or vacuum dry for 2 minutes) prior to addition of 50–100ml of ddhz0/ultrapure water or TE buffer.
After pour off supernatant, fast spin tube and suck dry residual in the tube before air dry. Usually no DNA or RNA pellet is visible at the bottom of the tube. Heat ddH2O/ultrapure water or TE buffer to 65°C before adding to sample to assist the DNA or RNA to dissolve in it.
Step 3 can be employed for single step purification of biomolecules after downstream application (PCR or RE digestion… etc.), add 5 volumes of DRoP3 (proceed to spinning and drying as in step 3) and reconstitute sample with ddH2O/ultrapure to initial volume.
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